Methods for enhancing efficacy of therapeutic immune cells

ABSTRACT

The present invention relates to a method of using a receptor (e.g., chimeric antigen receptor—CAR) that activates an immune response upon binding a cancer cell ligand in conjunction with a target-binding molecule that targets a protein or molecule for removal or neutralization to generate enhanced anti-cancer immune cells. The present invention also relates to engineered immune cells having enhanced therapeutic efficacy and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/943,400, filed Jul. 30, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/548,577, filed Aug. 3, 2017, now U.S. Pat. No.10,765,699, which is a 371 U.S. National Phase Application ofInternational Patent Cooperation Treaty Application PCT/SG2016/050063,filed Feb. 5, 2016, which claims benefit to U.S. Provisional ApplicationNo. 62/112,765, filed Feb. 6, 2015, and U.S. Provisional Application No.62/130,970, filed Mar. 10, 2015, the disclosures of which areincorporated herein by reference in their entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jul. 11, 2022, isnamed 62190_701_303 SL.xml and is 87,728 bytes in size.

BACKGROUND OF THE INVENTION

Immune cells can be potent and specific “living drugs”. Immune cellshave the potential to target tumor cells while sparing normal tissues;several clinical observations indicate that they can have majoranti-cancer activity. Thus, in patients receiving allogeneichematopoietic stem cell transplantation (HSCT), T-cell-mediatedgraft-versus-host disease (GvHD) (Weiden, P L et al., N. Engl. J. Med.1979; 300(19):1068-1073; Appelbaum, FR Nature, 2001; 411(6835):385-389;Porter, D L et al., N. Engl. J. Med. 1994; 330(2):100-106; Kolb, H J etal. Blood. 1995; 86(5):2041-2050; Slavin, S. et al., Blood. 1996;87(6):2195-2204), and donor natural killer (NK) cell alloreactivity(Ruggeri L, et al. Science. 2002; 295(5562):2097-2100; Giebel S, et al.Blood. 2003; 102(3):814-819; Cooley S, et al. Blood. 2010;116(14):2411-2419) are inversely related to leukemia recurrence. Besidesthe HSCT context, administration of antibodies that release T cells frominhibitory signals (Sharma P, et al., Nat Rev Cancer. 2011;11(11):805-812; Pardoll D M., Nat Rev Cancer. 2012; 12(4):252-264), orbridge them to tumor cells (Topp M S, et al. J. Clin. Oncol. 2011;29(18):2493-2498) produced major clinical responses in patients witheither solid tumors or leukemia. Finally, infusion ofgenetically-modified autologous T lymphocytes induced complete anddurable remission in patients with refractory leukemia and lymphoma(Maude S L, et al. N Engl J Med. 2014; 371(16):1507-1517).

Nevertheless, there is a significant need for improving immune celltherapy by broadening its applicability and enhancing its efficacy.

SUMMARY OF THE INVENTION

The present invention relates to engineered immune cells having enhancedtherapeutic efficacy for, e.g., cancer therapy. In certain embodiments,the present invention provides an engineered immune cell that comprisesa nucleic acid comprising a nucleotide sequence encoding an immuneactivating receptor, and a nucleic acid comprising a nucleotide sequenceencoding a target-binding molecule linked to a localizing domain.

In other embodiments, the present invention provides the use of anengineered immune cell that comprises a gene encoding an immuneactivating receptor, and a gene encoding a target-binding moleculelinked to a localizing domain for treating cancer, comprisingadministering a therapeutic amount of the engineered immune cell to asubject in need thereof.

In various embodiments, the present invention also provides a method forproducing an engineered immune cell, the method comprising introducinginto an immune cell a nucleic acid comprising a nucleotide sequenceencoding an immune activating receptor, and a nucleic acid comprising anucleotide sequence encoding a target-binding molecule linked to alocalizing domain, thereby producing an engineered immune cell.

In some embodiments, the engineered immune cells possess enhancedtherapeutic efficacy as a result of one or more of reducedgraft-versus-host disease (GvHD) in a host, reduced or elimination ofrejection by a host, extended survival in a host, reduced inhibition bythe tumor in a host, reduced self-killing in a host, reducedinflammatory cascade in a host, or sustained natural/artificialreceptor-mediated (e.g., CAR-mediated) signal transduction in a host.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIGS. 1A-1B is a schematic representation of a strategy employed in thepresent invention. FIG. 1A is an overall mechanism of CAR mediatedkilling of cancer cells. FIG. 1B shows the combined expression of CARwith different formats of compartment-directed scFv (an example of atarget-binding molecule linked to a localizing domain) and examples ofpossible targets. The CAR can be replaced by other receptors that canenhance immune cell capacity.

FIG. 2 is a schematic diagram of constructs containing scFv togetherwith domains that localize them to specific cellular compartments.Abbreviations: β2M, β-2 microglobulin; SP, signal peptide; VL, variablelight chain; VH, variable heavy chain; TM, transmembrane domain; HA,human influenza hemagglutinin. Additional constructs not listed in thefigure include membrane-bound (mb) myc EEKKMP, mb myc KKTN, mb myc YQRL,mb TGN38 cytoplasmic domain, mb myc RNIKCD, linker (20-amino acid) mbEEKKMP, as well as variants of constructs without signaling peptide andwith a varying number of amino acids in the CD8 transmembrane domain.The nucleotide sequence of the 10-amino acid linker isGGTGGTGGCGGCAGTGGTGGCGGTGGCTCA (SEQ ID NO: 61); the amino acid sequenceis GGGGSGGGGS (SEQ ID NO: 62). The nucleotide sequence of the 20-aminoacid linker is GGTGGTGGCGGCAGTGGTGGCGGTGGCTCAGGCGGTGGTGGCTCCGGTGGCGGTGGCTCT (SEQ ID NO: 63); the amino acid sequence is GGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 41). Various localization domains are indicated under theheading “Localization domains,” and depicts linkers in some examples, asindicated. The constructs “myc KDEL” and “PEST KDEL” show the use ofmore than one localization domains in a single construct.

FIGS. 3A-3C show downregulation of CD3/TCR in T cells by scFv targetingof CD3ε. FIG. 3A shows expression of surface CD3ε in Jurkat cells,transduced with either a retroviral vector containing green fluorescentprotein (GFP) only (“mock”) or a vector containing GFP plus differentconstructs as indicated Expression of CD3ε on the cell membrane wascompared to that of mock-transduced cells 1 week after transductionusing an anti-CD3 antibody conjugated to allophycocyanin (BDBiosciences). All comparisons were performed after gating onGFP-positive cells. FIG. 3B depicts similar experiments performed withperipheral blood T lymphocytes expanded with anti-CD3/CD28 beads(Lifesciences). Staining was performed 1 week after transduction. FIG.3C shows flow cytometry plots illustrating downregulation of membraneCD3ε in Jurkat cells after transduction with the constructs indicated.Dashed rectangles on the upper right quadrant of each plot encloseGFP+CD3+ cells.

FIG. 4 shows downregulation of CD3ε and TCRαβ on the cell membrane inJurkat T cells upon transduction with anti-CD3ε scFv-KDEL or -PEST, or-mb EEKKMP. Membrane marker expression was measured 1 week aftertransduction using an anti-CD3 antibody conjugated to allophycocyanin(BD Biosciences) or an anti-TCRαβ conjugated to phycoerythrin(Biolegend). Lines labeled “Control” represent labelling ofmock-transduced cells. Dashed vertical line represents the upper limitof staining obtained with an isotype-matched non-reactive antibody.

FIG. 5 shows that anti-scFv and CAR can be expressed simultaneously.Flow cytometric dot plots represent staining of Jurkat cells (top row)or peripheral blood lymphocytes (bottom row) with anti-CD3allophycocyanin antibody and goat-anti-mouse Fab2 biotin plusstreptavidin conjugated to phycoerythrin (to detect the CAR). Cells weretransduced with the anti-CD3 scFv-myc KDEL construct, theanti-CD19-4-1BB-CD3ζ construct, or both. After gating on GFP-positivecells, those transduced with anti-CD3 scFv-myc KDEL downregulated CD3(left column, bottom left quadrants) and those transduced with theanti-CD19-4-1BB-CD3ζ construct expressed the CAR (middle column, topright quadrants). A substantial proportion of cells transduced with bothconstructs were CD3-negative and CAR-positive (right column, top leftquadrants).

FIG. 6 illustrates that anti-CD19 CAR triggers T cell activation anddegranulation regardless of CD3/TCR downregulation. Jurkat cells weretransduced with the anti-CD3 scFv-myc KDEL construct, theanti-CD19-4-1BB-CD3ζ construct, or both. T cell activation anddegranulation was compared to that of mock-transduced cells. Cells wereco-cultured alone or with the CD19+ leukemia cell line OP-1 at a 1:1ratio. After 18 hours, expression of CD69 and CD25 was tested by flowcytometry using specific antibodies (from BD Biosciences); expression ofCD107a was tested after 6 hours (antibody from BD Biosciences). In thepresence of OP-1 cells, CD69 and CD25 expression in CAR-expressing cellsoccurred regardless of whether cells were also transduced with anti-CD3scFv-KDEL; no activation occurred in mock- or anti-CD3 scFv-myc KDELtransduced cells, or in the absence of OP-1 cells. CAR stimulationenhanced CD107 expression which was not affected by CD3/TCRdownregulation.

FIG. 7 shows that anti-CD19 CAR expressed in T cells causes T cellproliferation regardless of CD3/TCR downregulation. Peripheral blood Tlymphocytes were transduced with both the anti-CD3 scFv-myc KDELconstruct and the anti-CD19-4-1BB-CD3ζ construct. Transduced Tlymphocytes were co-cultured with OP-1 cells treated with Streck (Omaha,Nebr.) to inhibit their proliferation for the time indicated. Expansionof CD3-positive and CD3-negative T lymphocytes expressing the anti-CD19CAR was compared to that of mock-transduced T cells. Each symbol showsthe average cell count of two parallel cultures. CAR T cell expandedequally well regardless of CD3/TCR expression.

FIG. 8 shows expression of CD7 on the membrane of peripheral blood Tlymphocytes transduced with either a retroviral vector containing GFPonly (“mock”) or a vector containing GFP plus and anti-CD7 scFv-myc KDELconstruct. Expression of CD7 on the cell membrane was compared to thatof mock-transduced cells 1 week after transduction using an anti-CD7antibody conjugated to phycoerythrin (BD Biosciences). Dashed rectangleson the upper right quadrant of each plot enclose GFP+CD7+ cells.

FIG. 9 depicts the downregulation of HLA Class I in T cells by scFvtargeting of β2-microglobulin. Jurkat T cells were transduced withanti-β2M scFv-myc KDEL. Expression of HLA-ABC on the cell membrane wascompared to that of mock-transduced cells 1 week after transductionusing an anti-HLA-ABC antibody conjugated to phycoerythrin (BDBiosciences). Staining with an isotype-matched control antibody is alsoshown. Analysis was performed after gating on GFP-positive cells.

FIG. 10 depicts the downregulation of Killer Immunoglobulin-likeReceptor (KIR) 2DL1 and KIR2DL2/DL3 in human NK cells by scFv targetingof KIR2DL1 and KIR2DL2/DL3. NK cells, expanded ex vivo and selected forKIR2DL1 expression, were transduced with anti-KIR2DL1-KIR2DL2/DL3scFv-linker (20) AEKDEL or -EEKKMP. Expression of the corresponding MRon the cell membrane was compared to that of mock-transduced cells 8days after transduction using an anti-KIR2DL1 antibody conjugated toallophycocyanin (R&D Systems) or an anti-KIR2DL2/DL3 antibody conjugatedto phycoerythrin (BD Biosciences). Staining with an isotype-matchedcontrol antibody is also shown. Analysis was performed after gating onGFP-positive cells.

FIG. 11 depicts the downregulation of NKG2A in human NK cells by scFvtargeting. NK cells, expanded ex vivo, were transduced with anti-NKG2AscFv-EEKKMP. Expression of NKG2A on the cell membrane was compared tothat of mock-transduced cells 8 days after transduction using an NKG2Aantibody conjugated to phycoerythrin (Beckman Coulter). Staining with anisotype-matched control antibody is also shown. Analysis was performedafter gating on GFP-positive cells.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

In recent years, gains in knowledge about the molecular pathways thatregulate immune cells have been paralleled by a remarkable evolution inthe capacity to manipulate them ex vivo, including their expansion andgenetic engineering. It is now possible to reliably prepare highlysophisticated clinical-grade immune cell products in a timely fashion. Aprime example of how the anti-cancer activity of immune cells can bedirected and magnified by ex vivo cell engineering is the development ofchimeric antigen receptor (CAR) T cells (Eshhar, Z. et al., PNAS. 1993;90(2):720-724).

CARs are artificial multi-molecular proteins, which have been previouslydescribed (Geiger T L, et al., J Immunol. 1999; 162(10):5931-5939;Brentjens R J, et al., NatMed. 2003; 9(3):279-286; Cooper L J, et al.,Blood. 2003; 101(4):1637-1644). CARs comprise an extracellular domainthat binds to a specific target, a transmembrane domain, and acytoplasmic domain. The extracellular domain and transmembrane domaincan be derived from any desired source for such domains, as describedin, e.g., U.S. Pat. No. 8,399,645, incorporated by reference herein inits entirety. Briefly, a CAR may be designed to contain a single-chainvariable region (scFv) of an antibody that binds specifically to atarget. The scFv may be linked to a T-cell receptor (TCR)-associatedsignaling molecule, such as CD3ζ, via transmembrane and hinge domains.Ligation of scFv to the cognate antigen triggers signal transduction.Thus, CARs can instantaneously redirect cytotoxic T lymphocytes towardscancer cells and provoke tumor cell lysis (Eshhar, Z. et al., PNAS.1993; 90(2):720-724; Geiger T L, et al., J Immunol. 1999;162(10):5931-5939; Brentjens R J, et al., NatMed. 2003; 9(3):279-286;Cooper L J, et al., Blood. 2003; 101(4):1637-1644; Imai C, et al.,Leukemia. 2004; 18:676-684). Because CD3ζ signaling alone is notsufficient to durably activate T cells (Schwartz R H. Annu Rev Immunol.2003; 21:305-334; Zang X and Allison J P. Clin Cancer Res. 2007; 13(18Pt 1):5271-5279), co-stimulatory molecules such as CD28 and 4-1BB (orCD137) have been incorporated into CAR constructs to boost signaltransduction. This dual signaling design (“second generation CAR”) isuseful to elicit effective anti-tumor activity from T cells (Imai C, etal., Leukemia. 2004; 18:676-684; Campana D, et al., Cancer J. 2014;20(2):134-140).

A specific CAR, anti-CD19 CAR, containing both 4-1BB and CD3ζ has beendescribed in U.S. Pat. No. 8,399,645. Infusion of autologous T cellsexpressing an anti-CD19-4-1BB-CD3ζ CAR resulted in dramatic clinicalresponses in patients with chronic lymphocytic leukemia (CLL) (Porter DL, et al., Chimeric antigen receptor-modified T cells in chroniclymphoid leukemia; 2011: N Engl J Med. 2011; 365(8):725-733; Kalos M, etal., SciTranslMed. 2011; 3(95):95ra73), and acute lymphoblastic leukemia(ALL) (Grupp S A, et al., N Engl J Med. 2013; 368(16):1509-1518; Maude SL, et al., N Engl J Med. 2014; 371(16):1507-1517). These studies, andstudies with CARs bearing different signaling modules (Till B G, et al.,Blood. 2012; 119(17):3940-3950; Kochenderfer J N, et al., Blood. 2012;119(12):2709-2720; Brentjens R J, et al., Blood. 2011;118(18):4817-4828; Brentjens R J, et al., Sci Transl Med. 2013;5(177):177ra138), provide a convincing demonstration of the clinicalpotential of this technology, and of immunotherapy in general.

The methods described herein enable rapid removal or inactivation ofspecific proteins in immune cells redirected by a natural or artificialreceptor, e.g., CARs, thus broadening the application potential andsignificantly improving the function of the engineered cells. The methodrelies, in part, on a single construct or multiple constructs containingan immune activating receptor, e.g., a CAR (which comprises anextracellular domain (e.g., an scFv) that binds to a specific target, atransmembrane domain, and a cytoplasmic domain) together with atarget-binding molecule that binds a target (e.g., protein) to beremoved or neutralized; the target-binding molecule is linked to adomain (i.e., localizing domain) that directs it to specific cellularcompartments, such as the Golgi or endoplasmic reticulum, theproteasome, or the cell membrane, depending on the application. Forsimplicity, a target-binding molecule linked to a localizing domain (LD)is sometimes referred to herein as “LD-linked target-binding molecule.”

As will be apparent from the teachings herein, a variety of immuneactivating receptors may be suitable for the methods of the presentinvention. That is, any receptor that comprises a molecule that, uponbinding (ligation) to a ligand (e.g., peptide or antigen) expressed on acancer cell, is capable of activating an immune response may be usedaccording to the present methods. For example, as described above, theimmune activating receptor can be a chimeric antigen receptor (CAR);methods for designing and manipulating a CAR is known in the art (see,Geiger T L, et al., J Immunol. 1999; 162(10):5931-5939; Brentjens R J,et al., NatMed. 2003; 9(3):279-286; Cooper L J, et al., Blood. 2003;101(4):1637-1644). Additionally, receptors with antibody-bindingcapacity can be used (e.g., CD16-4-1BB-CD3zeta receptor—Kudo K, et al.Cancer Res. 2014; 74(1):93-103), which are similar to CARs, but with thescFv replaced with an antibody-binding molecule (e.g., CD16, CD64,CD32). Further, T-cell receptors comprising T-cell receptor alpha andbeta chains that bind to a peptide expressed on a tumor cell in thecontext of the tumor cell HLA can also be used according to the presentmethods. In addition, other receptors bearing molecules that activate animmune response by binding a ligand expressed on a cancer cell can alsobe used—e.g., NKG2D-DAP10-CD3zeta receptor, which binds to NKG2D ligandexpressed on tumor cells (see, e.g., Chang Y H, et al., Cancer Res.2013; 73(6):1777-1786). All such suitable receptors collectively, asused herein, are referred to as an “immune activating receptor” or a“receptor that activates an immune response upon binding a cancer cellligand.” Therefore, an immune activating receptor having a moleculeactivated by a cancer cell ligand can be expressed together with aLD-linked target-binding molecule according to the present methods.

The present methods significantly expand the potential applications ofimmunotherapies based on the infusion of immune cells redirected byartificial receptors. The method described is practical and can beeasily incorporated in a clinical-grade cell processing. For example, asingle bicistronic construct containing, e.g., a CAR and a LD-linkedtarget-binding molecule, e.g., scFv-myc KDEL (or PEST or transmembrane)can be prepared by inserting an internal ribosomal entry site (IRES) ora 2A peptide-coding region site between the 2 cDNAs encoding the CAR andthe LD-linked target-binding molecule. The design of tricistronicdelivery systems to delete more than one target should also be feasible.Alternatively, separate transductions of the 2 genes (simultaneously orsequentially) could be performed. In the context of cancer cell therapy,the CAR could be replaced by an antibody-binding signaling receptor(Kudo K, et al., Cancer Res. 2014; 74(1):93-103), a T-cell receptordirected against a specific HLA-peptide combination, or any receptoractivated by contact with cancer cells (Chang Y H, et al., Cancer Res.2013; 73(6):1777-1786). The results of the studies described herein withsimultaneous anti-CD19-4-1BB-CD3ζ CAR and anti-CD3ε scFv-KDELdemonstrate that the signaling capacity of the CAR was not impaired.

Both the anti-CD3ε scFv-KDEL (and -PEST) tested herein stablydownregulate CD3 as well as TCR expression. Residual CD3+ T cells couldbe removed using CD3 beads, an approach that is also available in aclinical-grade format. The capacity to generate CD3/TCR-negative cellsthat respond to CAR signaling represents an important advance. Clinicalstudies with CAR T cells have generally been performed using autologousT cells. Thus, the quality of the cell product varies from patient topatient and responses are heterogeneous. Infusion of allogeneic T cellsis currently impossible as it has an unacceptably high risk ofpotentially fatal GvHD, due to the stimulation of the endogenous TCR bythe recipient's tissue antigens. Downregulation of CD3/TCR opens thepossibility of infusing allogeneic T cells because lack of endogenousTCR eliminates GvHD capacity. Allogeneic products could be prepared withthe optimal cellular composition (e.g., enriched in highly cytotoxic Tcells, depleted of regulatory T cells, etc.) and selected so that thecells infused have high CAR expression and functional potency. Moreover,fully standardized products could be cryopreserved and be available foruse regardless of the patient immune cell status and his/her fitness toundergo apheresis or extensive blood draws. Removal of TCR expressionhas been addressed using gene editing tools, such as nucleases (TorikaiH, et al. Blood, 2012; 119(24):5697-5705). Although this is an effectiveapproach, it is difficult to implement in a clinical setting as itrequires several rounds of cell selection and expansion, with prolongedculture. The methods described herein have considerable practicaladvantages.

Additionally, a LD-linked target-binding molecule (e.g., scFv-myc KDEL,scFv-EEKKMP or scFv-PEST, wherein scFv targets a specificprotein/molecule) can be used according to the present invention todelete HLA Class I molecules, reducing the possibility of rejection ofallogeneic cells. While infusion of allogeneic T cells is a future goalof CAR T cell therapy, infusion of allogeneic natural killer (NK) cellsis already in use to treat patients with cancer. A key factor thatdetermines the success of NK cell-based therapy is that NK cells mustpersist in sufficient numbers to achieve an effector: target ratiolikely to produce tumor cytoreduction (Miller J S. Hematology Am SocHematol Educ Program. 2013; 2013:247-253). However, when allogeneiccells are infused, their persistence is limited. Immunosuppressivechemotherapy given to the patient allows transient engraftment of theinfused NK cells but these are rejected within 2-4 weeks of infusion(Miller J S, et al. Blood. 2005; 105:3051-3057; Rubnitz J E, et al., JClin Oncol. 2010; 28(6):955-959). Contrary to organ transplantation,continuing immunosuppression is not an option because immunosuppressivedrugs also suppress NK cell function. Because rejection is primarilymediated by recognition of HLA Class I molecules by the recipient'sCD8+T lymphocytes, removing HLA Class I molecules from the infused NKcells (or T cells) will diminish or abrogate the rejection rate, extendthe survival of allogeneic cells, and hence their anti-tumor capacity.

Furthermore, a LD-linked target-binding molecule can be used accordingto the present invention to target inhibitory receptors. Specifically,administration of antibodies that release T cells from inhibitorysignals such as anti-PD1 or anti-CTLA-4 have produced dramatic clinicalresponses (Sharma P, et al., Nat Rev Cancer. 2011; 11(11):805-812;Pardoll D M. Nat Rev Cancer. 2012; 12(4):252-264). CAR-T cells,particularly those directed against solid tumors, might be inhibited bysimilar mechanisms. Thus, expression of a target-binding molecule (e.g.,scFv or ligands) against PD1, CTLA-4, Tim3 or other inhibitory receptorswould prevent the expression of these molecules (if linked to, e.g.,KDEL (SEQ ID NO: 4), EEKKMP (SEQ ID NO: 64) or PEST motifSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7)) or preventbinding of the receptors to their ligands (if linked to a transmembranedomain) and sustain CAR-mediated signal transduction. In NK cells,examples of inhibitory receptors include killer immunoglobulin-likereceptors (KIRs) and NKG2A (Vivier E, et al., Science, 2011;331(6013):44-49).

The methods of the present invention also enable targeting of a greaternumber of targets amenable for CAR-directed T cell therapy. One of themain limitations of CAR-directed therapy is the paucity of specificantigens expressed by tumor cells. In the case of hematologicmalignancies, such as leukemias and lymphomas, molecules which are notexpressed in non-hematopoietic cells could be potential targets butcannot be used as CAR targets because they are also expressed on T cellsand/or NK cells. Expressing such CARs on immune cells would likely leadto the demise of the immune cells themselves by a “fratricidal”mechanism, nullifying their anti-cancer capacity. If the target moleculecan be removed from immune cells without adverse functional effects,then the CAR with the corresponding specificity can be expressed. Thisopens many new opportunities to target hematologic malignancies.Examples of the possible targets include CD38 expressed in multiplemyeloma, CD7 expressed in T cell leukemia and lymphoma, Tim-3 expressedin acute leukemia, CD30 expressed in Hodgkin disease, CD45 and CD52expressed in all hematologic malignancies. These molecules are alsoexpressed in a substantial proportion of T cells and NK cells.

Moreover, it has been shown that secretion of cytokines by activatedimmune cells triggers cytokine release syndrome and macrophageactivation syndrome, presenting serious adverse effects of immune celltherapy (Lee D W, et al., Blood. 2014; 124(2):188-195). Thus, theLD-linked target-binding molecule can be used according to the presentinvention to block cytokines such as IL-6, IL-2, IL-4, IL-7, IL-10,IL-12, IL-15, IL-18, IL-21, IL-27, IL-35, interferon (IFN)-γ, IFN-β,IFN-α, tumor necrosis factor (TNF)-α, and transforming growth factor(TGF)-β, which may contribute to such inflammatory cascade.

Accordingly, in one embodiment, the present invention relates to anengineered immune cell that comprises a nucleic acid comprising anucleotide sequence encoding an immune activating receptor, and anucleic acid comprising a nucleotide sequence encoding a target-bindingmolecule linked to a localizing domain.

As used herein, an “engineered” immune cell includes an immune cell thathas been genetically modified as compared to a naturally-occurringimmune cell. For example, an engineered T cell produced according to thepresent methods carries a nucleic acid comprising a nucleotide sequencethat does not naturally occur in a T cell from which it was derived. Insome embodiments, the engineered immune cell of the present inventionincludes a chimeric antigen receptor (CAR) and a target-binding moleculelinked to a localizing domain (LD-linked target-binding molecule). In aparticular embodiment, the engineered immune cell of the presentinvention includes an anti-CD19-4-1BB-CD3ζ CAR and an anti-CD3 scFvlinked to a localizing domain.

In certain embodiments, the engineered immune cell is an engineered Tcell, an engineered natural killer (NK) cell, an engineered NK/T cell,an engineered monocyte, an engineered macrophage, or an engineereddendritic cell.

In certain embodiments, an “immune activating receptor” as used hereinrefers to a receptor that activates an immune response upon binding acancer cell ligand. In some embodiments, the immune activating receptorcomprises a molecule that, upon binding (ligation) to a ligand (e.g.,peptide or antigen) expressed on a cancer cell, is capable of activatingan immune response. In one embodiment, the immune activating receptor isa chimeric antigen receptor (CAR); methods for designing andmanipulating a CAR are known in the art. In other embodiments, theimmune activating receptor is an antibody-binding receptor, which issimilar to a CAR, but with the scFv replaced with an antibody-bindingmolecule (e.g., CD16, CD64, CD32) (see e.g., CD16-4-1BB-CD3zetareceptor—Kudo K, et al. Cancer Res. 2014; 74(1):93-103). In variousembodiments, T-cell receptors comprising T-cell receptor alpha and betachains that bind to a peptide expressed on a tumor cell in the contextof the tumor cell HLA can also be used according to the present methods.In certain embodiments, other receptors bearing molecules that activatean immune response by binding a ligand expressed on a cancer cell canalso be used—e.g., NKG2D-DAP10-CD3zeta receptor, which binds to NKG2Dligand expressed on tumor cells (see, e.g., Chang Y H, et al., CancerRes. 2013; 73(6):1777-1786). All such suitable receptors capable ofactivating an immune response upon binding (ligation) to a ligand (e.g.,peptide or antigen) expressed on a cancer cell are collectively referredto as an “immune activating receptor.” As would be appreciated by thoseof skill in the art, an immune activating receptor need not contain anantibody or antigen-binding fragment (e.g., scFv); rather the portion ofthe immune activating receptor that binds to a target molecule can bederived from, e.g., a receptor in a receptor-ligand pair, or a ligand ina receptor-ligand pair.

In certain aspects, the immune activating receptor binds to moleculesexpressed on the surface of tumor cells, including but not limited to,CD20, CD22, CD33, CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD52, CD38, CS-1,TIM3, CD123, mesothelin, folate receptor, HER2-neu, epidermal-growthfactor receptor, and epidermal growth factor receptor. In someembodiments, the immune activating receptor is a CAR (e.g.,anti-CD19-4-1BB-CD3ζ CAR). In certain embodiments, the immune activatingreceptor comprises an antibody or antigen-binding fragment thereof(e.g., scFv) that binds to molecules expressed on the surface of tumorcells, including but not limited to, CD20, CD22, CD33, CD2, CD3, CD4,CD5, CD7, CD8, CD45, CD52, CD38, CS-1, TIM3, CD123, mesothelin, folatereceptor, HER2-neu, epidermal-growth factor receptor, and epidermalgrowth factor receptor. Antibodies to such molecules expressed on thesurface of tumor cells are known and available in the art. By way ofexample, antibodies to CD3 and CD7 are commercially available and knownin the art. Such antibodies, as well as fragments of antibodies (e.g.,scFv) derived therefrom, can be used in the present invention, asexemplified herein. Further, methods of producing antibodies andantibody fragments against a target protein are well-known and routinein the art.

The transmembrane domain of an immune activating receptor according tothe present invention (e.g., CAR) can be derived from a single-passmembrane protein, including, but not limited to, CD8α, CD8β, 4-1BB,CD28, CD34, CD4, FcεRIγ, CD16 (e.g., CD16A or CD16B), OX40, CD3ζ, CD3ε,CD3γ, CD3δ, TCRα, CD32 (e.g., CD32A or CD32B), CD64 (e.g., CD64A, CD64B,or CD64C), VEGFR2, FAS, and FGFR2B. In some examples, the membraneprotein is not CD8α. The transmembrane domain may also be anon-naturally occurring hydrophobic protein segment.

The hinge domain of the immune activating receptor (e.g., CAR) can bederived from a protein such as CD8α, or IgG. The hinge domain can be afragment of the transmembrane or hinge domain of CD8α, or anon-naturally occurring peptide, such as a polypeptide consisting ofhydrophilic residues of varying length, or a (GGGGS)_(n)(SEQ ID NO: 8)polypeptide, in which n is an integer of, e.g., 3-12, inclusive.

The signaling domain of the immune activating receptor (e.g., CAR) canbe derived from CD3ζ, FcεRIγ, DAP10, DAP12 or other molecules known todeliver activating signals in immune cells. At least one co-stimulatorysignaling domain of the receptor can be a co-stimulatory molecule suchas 4-1BB (also known as CD137), CD28, CD28LL-*GG variant, OX40, ICOS,CD27, GITR, HVEM, TIM1, LFA1, or CD2. Such molecules are readilyavailable and known in the art.

As would be appreciated by those of skill in the art, the components ofan immune activating receptor can be engineered to comprise a number offunctional combinations, as described herein, to produce a desiredresult. Using the particular CAR anti-CD19-4-1BB-CD3ζ as an example, theantibody (e.g., or antigen-binding fragment thereof such as an scFv)that binds a molecule can be substituted for an antibody that bindsdifferent molecule, as described herein (e.g., anti-CD20, anti-CD33,anti-CD123, etc., instead of anti-CD19). In other embodiments, theco-stimulatory molecule (4-1BB in this specific example) can also bevaried with a different co-stimulatory molecule, e.g., CD28. In someembodiments, the stimulatory molecule (CD3ζ in this specific example),can be substituted with another known stimulatory molecule. In variousembodiments, the transmembrane domain of the receptor can also be variedas desired. The design, production, and testing for functionality ofsuch immune activating receptors can be readily determined by those ofskill in the art. Similarly, the design, delivery into cells andexpression of nucleic acids encoding such immune activating receptorsare readily known and available in the art.

As used herein, the term “nucleic acid” refers to a polymer comprisingmultiple nucleotide monomers (e.g., ribonucleotide monomers ordeoxyribonucleotide monomers). “Nucleic acid” includes, for example,genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acidmolecules can be naturally occurring, recombinant, or synthetic. Inaddition, nucleic acid molecules can be single-stranded, double-strandedor triple-stranded. In some embodiments, nucleic acid molecules can bemodified. In the case of a double-stranded polymer, “nucleic acid” canrefer to either or both strands of the molecule.

The term “nucleotide sequence,” in reference to a nucleic acid, refersto a contiguous series of nucleotides that are joined by covalentlinkages, such as phosphorus linkages (e.g., phosphodiester, alkyl andaryl-phosphonate, phosphorothioate, phosphotriester bonds), and/ornon-phosphorus linkages (e.g., peptide and/or sulfamate bonds). Incertain embodiments, the nucleotide sequence encoding, e.g., atarget-binding molecule linked to a localizing domain is a heterologoussequence (e.g., a gene that is of a different species or cell typeorigin).

The terms “nucleotide” and “nucleotide monomer” refer to naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, as well asnon-naturally occurring derivatives and analogs thereof. Accordingly,nucleotides can include, for example, nucleotides comprising naturallyoccurring bases (e.g., adenosine, thymidine, guanosine, cytidine,uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, ordeoxycytidine) and nucleotides comprising modified bases known in theart.

As will be appreciated by those of skill in the art, in some aspects,the nucleic acid further comprises a plasmid sequence. The plasmidsequence can include, for example, one or more sequences selected fromthe group consisting of a promoter sequence, a selection markersequence, and a locus-targeting sequence.

As used herein, the gene encoding a target-binding molecule linked to alocalizing domain is sometimes referred to as “LD-linked target-bindingmolecule.”

In certain embodiments, the target-binding molecule is an antibody orantigen-binding fragment thereof. As used herein, “antibody” means anintact antibody or antigen-binding fragment of an antibody, including anintact antibody or antigen-binding fragment that has been modified orengineered, or that is a human antibody. Examples of antibodies thathave been modified or engineered are chimeric antibodies, humanizedantibodies, multiparatopic antibodies (e.g., biparatopic antibodies),and multispecific antibodies (e.g., bispecific antibodies). Examples ofantigen-binding fragments include Fab, Fab′, F(ab′)₂, Fv, single chainantibodies (e.g., scFv), minibodies and diabodies.

A “Fab fragment” comprises one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the CH2 andCH3 domains of an antibody. The two heavy chain fragments are heldtogether by two or more disulfide bonds and by hydrophobic interactionsof the CH3 domains.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the VH domain and the CH1 domain and also the regionbetween the CH1 and CH2 domains, such that an interchain disulfide bondcan be formed between the two heavy chains of two Fab′ fragments to forma F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)′ domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′), fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

In a particular embodiment, the target-binding molecule is single-chainFv antibody (“scFv antibody”). scFv refers to antibody fragmentscomprising the VH and VL domains of an antibody, wherein these domainsare present in a single polypeptide chain. Generally, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainswhich enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Pluckthun (1994) The Pharmacology OfMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315. See also, PCT Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. By way of example,the linker between the VH and VL domains of the scFvs disclosed hereincomprise, e.g., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGSGGGGS(SEQ ID NO: 43). As would be appreciated by those of skill in the art,various suitable linkers can be designed and tested for optimalfunction, as provided in the art, and as disclosed herein.

The scFv that is part of the LD-linked target-binding molecule is notnecessarily the same as the scFv that occurs in the context of, e.g., achimeric antigen receptor (CAR) or a similar antibody-binding signalingreceptor. In some embodiments, the scFv that is part of the LD-linkedtarget-binding molecule is the same as the scFv that occurs in thecontext of, e.g., a chimeric antigen receptor (CAR) or a similarantibody-binding signaling receptor.

In some embodiments, the nucleic acid comprising a nucleotide sequenceencoding a target-binding molecule (e.g., an scFv in the context of aLD-linked target-binding molecule) comprises one or more sequences thathave at least 80%, at least 85%, at least 88%, at least 90%, at least92%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to any one or more of SEQID NOs: 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, or 39.

The term “sequence identity” means that two nucleotide or amino acidsequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least, e.g., 70% sequenceidentity, or at least 80% sequence identity, or at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity or more. For sequence comparison, typically one sequence actsas a reference sequence (e.g., parent sequence), to which test sequencesare compared. When using a sequence comparison algorithm, test andreference sequences are input into a computer, subsequence coordinatesare designated, if necessary, and sequence algorithm program parametersare designated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In certain embodiments, the antibody (e.g., scFv) comprises VH and VLhaving amino acid sequences set forth in SEQ ID NO: 12 and 13,respectively; SEQ ID NO: 16 and 17, respectively; SEQ ID NO: 20 and 21,respectively; SEQ ID NO: 24 and 25, respectively; SEQ ID NO: 28 and 29,respectively; SEQ ID NO: 32 and 33, respectively; or SEQ ID NO: 36 and37, respectively. In some embodiments, the antibody (e.g., scFv)comprises VH and VL having sequence that each have at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH and VL sequences set forth in SEQ ID NO: 12 and 13, respectively;SEQ ID NO: 16 and 17, respectively; SEQ ID NO: 20 and 21, respectively;SEQ ID NO: 24 and 25, respectively; SEQ ID NO: 28 and 29, respectively;SEQ ID NO: 32 and 33, respectively; or SEQ ID NO: 36 and 37,respectively.

A “diabody” is a small antibody fragment with two antigen-binding sites.The fragments comprise a heavy chain variable region (VH) connected to alight chain variable region (VL) in the same polypeptide chain (VH-VL orVL-VH). By using a linker that is too short to allow pairing between thetwo domains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described in, e.g., patent documents EP 404,097; WO93/11161; and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

In certain embodiments, the antibody is a triabody or a tetrabody.Methods of designing and producing triabodies and tetrabodies are knownin the art. See, e.g., Todorovska et al., J. Immunol. Methods248(1-2):47-66, 2001.

A “domain antibody fragment” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore VH regions are covalently joined with a peptide linker to create abivalent domain antibody fragment. The two VH regions of a bivalentdomain antibody fragment may target the same or different antigens.

In some embodiments, the antibody is modified or engineered. Examples ofmodified or engineered antibodies include chimeric antibodies,multiparatopic antibodies (e.g., biparatopic antibodies), andmultispecific antibodies (e.g., bispecific antibodies).

As used herein, “multiparatopic antibody” means an antibody thatcomprises at least two single domain antibodies, in which at least onesingle domain antibody is directed against a first antigenic determinanton an antigen and at least one other single domain antibody is directedagainst a second antigenic determinant on the same antigen. Thus, forexample, a “biparatopic” antibody comprises at least one single domainantibody directed against a first antigenic determinant on an antigenand at least one further single domain antibody directed against asecond antigenic determinant on the same antigen.

As used herein, “multispecific antibody” means an antibody thatcomprises at least two single domain antibodies, in which at least onesingle domain antibody is directed against a first antigen and at leastone other single domain antibody is directed against a second antigen(different from the first antigen). Thus, for example, a “bispecific”antibody is one that comprises at least one single domain antibodydirected against a first antigen and at least one further single domainantibody directed against a second antigen, e.g., different from thefirst antigen.

In some embodiments, the antibodies disclosed herein are monoclonalantibodies, e.g., murine monoclonal antibodies. Methods of producingmonoclonal antibodies are known in the art. See, for example, Pluckthun(1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburgand Moore eds. Springer-Verlag, New York, pp. 269-315.

In various embodiments, the target-binding molecule in the context of aLD-linked target-binding molecule is a receptor or a ligand that bindsto a target molecule. For example, that target-binding molecule can be aligand that binds PD-1 (e.g., PD-L1 or PD-L2). Thus, as would beappreciated by those of skill in the art, the target-binding moleculecan be an antibody, or a ligand/receptor that binds a target molecule.

As used herein, “linked” in the context of a LD-linked target-bindingmolecule refers to a gene encoding a target-binding molecule directly inframe (e.g., without a linker) adjacent to one or more genes encodingone or more localizing domains. Alternatively, the gene encoding atarget-binding molecule may be connected to one or more gene encodingone or more localizing domains through a linker sequence, as describedherein. Various suitable linkers known in the art can be used to tetherthe target-binding molecule to a localizing domain. For example,non-naturally occurring peptides, such as a polypeptide consisting ofhydrophilic residues of varying length, or a (GGGGS)_(n) (SEQ ID NO: 8)polypeptide, in which n is an integer of, e.g., 3-12, inclusive, can beused according to the present invention. In particular embodiments, thelinker comprises, e.g., GGGGSGGGGS (SEQ ID NO: 62). In some embodiments,the linker comprises, e.g., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41). Invarious embodiments, peptide linkers having lengths of about 5 to about100 amino acids, inclusive, can be used in the present invention. Incertain embodiments, peptide linkers having lengths of about 20 to about40 amino acids, inclusive, can be used in the present invention. In someembodiments, peptide linkers having lengths of at least 5 amino acids,at least 10 amino acids, at least 15 amino acids, at least 20 aminoacids, at least 25 amino acids, at least 30 amino acids, at least 35amino acids, or at least 40 amino acids can be used in the presentinvention. As would be appreciated by those of skill in the art, suchlinker sequences as well as variants of such linker sequences are knownin the art. Methods of designing constructs that incorporate linkersequences as well as methods of assessing functionality are readilyavailable to those of skill in the art.

In certain embodiments, the LD-linked target-binding molecule binds to atarget expressed on the surface of an immune cell. In some embodiments,the LD-linked target-binding molecule inhibits the activity or functionof the target molecule. By way of example, as disclosed herein, theLD-linked target-binding molecule can be designed to bind to, e.g., CD3,CD7, CD45, hB2MG, KIR2DL1, KIR2DL2/DL3, or NKG2A, thereby downregulatingthe cell surface expression of such molecules. Downregulation of suchmolecules can be achieved through, for example, localizing/targeting themolecules for degradation and/or internalization. In other embodiments,the LD-linked target-binding molecule renders the target inactive (e.g.,the target can no longer interact and/or bind to its cognate ligand orreceptor).

In some embodiments, the engineered immune cells of the presentinvention have enhanced therapeutic efficacy. As used herein, “enhancedtherapeutic efficacy” refers to one or more of reduced graft-versus-hostdisease (GvHD) in a host, reduced or elimination of rejection by a host,extended survival in a host, reduced inhibition by the tumor in a host,reduced self-killing in a host, reduced inflammatory cascade in a host,or sustained CAR-mediated signal transduction in a host.

In certain embodiments of the present invention, the target-bindingmolecule in the context of a LD-linked target-binding molecule binds toa molecule in a CD3/T-cell receptor (TCR) complex, a cytokine, a humanleukocyte antigen (HLA) Class I molecule, or a receptor thatdownregulates immune response.

In certain embodiments, a molecule in a CD3/TCR complex can be CD3ε,TCRα, TCRβ, TCRγ, TCRδ, CD3δ, CD3γ, or CD3ζ. In a particular embodiment,the molecule is CD3ε.

In another embodiment, the HLA Class I molecule is beta-2 microglobulin,α1-microglobulin, α2-microglobulin, or α3-microglobulin.

In other embodiments, a receptor that downregulates immune response isselected from, e.g., PD-1, CTLA-4, Tim3, killer immunoglobulin-likereceptors (KIRs—e.g., KIR2DL1 (also known as CD158a), KIR2DL2/DL3 (alsoknown as CD158b)), CD94 or NKG2A (also known as CD159a), proteintyrosine phosphatases such as Src homology region 2 domain-containingphosphatase (SHP)-1 and SHP-2. Thus, such receptors can be targeted bymoiety LD-linked target-binding molecule, as described herein.

In various embodiments, examples of cytokines that can be targeted withmoiety LD-linked target-binding molecule include, e.g., interleukin(IL)-6, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27,IL-35, interferon (IFN)-γ, IFN-β, IFN-α, tumor necrosis factor (TNF)-α,or transforming growth factor (TGF)-βα.

In a further aspect, the LD-linked target-binding molecule binds to amolecule selected from, e.g., CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45,CD52, or CD127.

Methods of producing antibodies and antibody fragments thereof againstany target protein are well-known and routine in the art. Moreover, asexemplified herein, commercially available antibodies to varioustargets, e.g., CD3 and CD7 can be used to generate a LD-linkedtarget-binding molecule, as exemplified herein. Antibodies known in theart, as well as fragments of antibodies (e.g., scFv) derived therefrom,can be used in the present invention, as exemplified herein.

In other aspects, the localizing domain of the LD-linked target-bindingmolecule comprises an endoplasmic reticulum (ER) retention sequence KDEL(SEQ ID NO: 4), or other ER or Golgi retention sequences such as KKXX(SEQ ID NO: 9), KXD/E (SEQ ID NO: 10) (where X can be any amino acid—seeGao C, et al., Trends in Plant Science 19: 508-515, 2014) and YQRL (SEQID NO: 11) (see Zhan J, et al., Cancer Immunol Immunother 46:55-60,1998); a proteosome targeting sequence that comprises, e.g., “PEST”motif-SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7); and/or asequence that targets the target-binding molecule to the cell membrane,such as the CD8a transmembrane domain, or the transmembrane of anothersingle-pass membrane protein, as described herein (e.g., CD8α, CD8β,4-1BB, CD28, CD34, CD4, FcεRIγ, CD16 (such as CD16A or CD16B), OX40,CD3λ, CD3ε, CD3γ, CD3δ, TCRα, CD32 (such as CD32A or CD32B), CD64 (suchas CD64A, CD64B, or CD64C), VEGFR2, FAS, or FGFR2B). Examples ofparticular localizing domains (sequences) exemplified herein are shownin FIG. 2 . Various other localizing sequences are known and availablein the art.

As shown in FIG. 2 , the LD-linked target-binding molecules of thepresent invention can comprise one or more localizing domains. Forexample, the LD-linked target-binding molecule can have at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or at least tenlocalizing domains linked together. When more than one localizing domainis used in a given LD-linked target-binding molecule, each localizingdomain can be linked with or without any intervening linker. By way ofexample, as shown in FIG. 2 , localization domains CD8 TM, PEST motif,and EEKKMP can be used in a single LD-linked target-binding molecule.While this particular construct shows the localization domains withoutany intervening linkers, various intervening linkers can be incorporatedbetween some or all of the localization domains. Other examples areshown in FIG. 2 .

As would be appreciated by those of skill in the art, the immuneactivating receptor and/or the LD-linked target-binding molecule can bedesigned to bind to the targets disclosed herein, as well as variants ofthe targets disclosed herein. By way of example, an immune activatingreceptor and/or the LD-linked target-binding molecule can be designed tobind to a molecule in a CD3/TCR complex, or a naturally-occurringvariant molecule thereof. Such naturally-occurring variants can have thesame function as the wild-type form of the molecule. In otherembodiments, the variant can have a function that is altered relative tothe wild-type form of the molecule (e.g., confers a diseased state).

As would be appreciated by those of skill in the art, the variouscomponents of the LD-linked target-binding molecule constructs shown inFIG. 2 can be substituted in different combinations (e.g., to contain adifferent linker, different localizing sequence, different scFv, etc.),so long as the combination produces a functional LD-linkedtarget-binding molecule. Methods of assessing functionality for aparticular construct are within the ambit of those of skill in the art,as disclosed herein.

In further aspects, the present invention relates to the use of anengineered immune cell that comprises a nucleic acid comprising anucleotide sequence encoding an immune activating receptor, and anucleic acid comprising a nucleotide sequence encoding a target-bindingmolecule (e.g., scFv) linked to a localizing domain for treating cancer,comprising administering a therapeutic amount of the engineered immunecell to a subject in need thereof.

In another aspect, the present invention relates to the use of anengineered immune cell that comprises a nucleic acid comprising anucleotide sequence encoding a chimeric antigen receptor (CAR) and anucleic acid comprising a nucleotide sequence encoding a single-chainvariable fragment (scFv) linked to a localizing domain for treatingcancer, comprising administering a therapeutic amount of the engineeredimmune cell to a subject in need thereof.

In other aspects, the present invention relates to the use of anengineered immune cell that comprises a nucleic acid comprising anucleotide sequence encoding an immune activating receptor, and anucleic acid comprising a nucleotide sequence encoding a target-bindingmolecule (e.g., scFv) linked to a localizing domain for treating anautoimmune disorder, comprising administering a therapeutic amount ofthe engineered immune cell to a subject in need thereof.

In other aspects, the present invention also relates to the use of anengineered immune cell that comprises a nucleic acid comprising anucleotide sequence encoding an immune activating receptor, and anucleic acid comprising a nucleotide sequence encoding a target-bindingmolecule (e.g., scFv) linked to a localizing domain for treating aninfectious disease, comprising administering a therapeutic amount of theengineered immune cell to a subject in need thereof.

In various embodiments, the immune activating receptor is a CAR (e.g.,anti-CD19-4-1BB-CD3ζ CAR).

In other embodiments, the single-chain variable fragment (scFv) linkedto a localizing domain is selected from any one or more constructs shownin FIG. 2 .

In some aspects, the engineered immune cell is administered by infusioninto the subject. Methods of infusing immune cells (e.g., allogeneic orautologous immune cells) are known in the art. A sufficient number ofcells are administered to the recipient in order to ameliorate thesymptoms of the disease. Typically, dosages of 10⁷ to 10¹⁰ cells areinfused in a single setting, e.g., dosages of 10⁹ cells. Infusions areadministered either as a single 10⁹ cell dose or divided into several10⁹ cell dosages. The frequency of infusions can be every 3 to 30 daysor even longer intervals if desired or indicated. The quantity ofinfusions is generally at least 1 infusion per subject and preferably atleast 3 infusions, as tolerated, or until the disease symptoms have beenameliorated. The cells can be infused intravenously at a rate of 50-250ml/hr. Other suitable modes of administration include intra-arterialinfusion, direct injection into tumor and/or perfusion of tumor bedafter surgery, implantation at the tumor site in an artificial scaffold,intrathecal administration, and intraocular administration. Methods ofadapting the present invention to such modes of delivery are readilyavailable to one skilled in the art.

In certain aspects, the cancer to be treated is a solid tumor or ahematologic malignancy. Examples of hematologic malignancies includeacute myeloid leukemia, chronic myelogenous leukemia, myelodysplasia,acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiplemyeloma, Hodgkin and non-Hodgkin lymphoma. Examples of solid tumorsinclude lung cancer, melanoma, breast cancer, prostate cancer, coloncancer, renal cell carcinoma, ovarian cancer, pancreatic cancer,hepatocellular carcinoma, neuroblastoma, rhabdomyosarcoma, brain tumor.

In another embodiment, the present invention relates to a method forproducing an engineered immune cell of the present invention, comprisingintroducing into an immune cell a nucleic acid comprising a nucleotidesequence encoding an immune activating receptor, and a nucleic acidcomprising a nucleotide sequence encoding a target-binding moleculelinked to a localizing domain, thereby producing an engineered immunecell.

In certain embodiments, the nucleic acid comprising a nucleotidesequence is introduced into an immune cell ex vivo. In otherembodiments, the nucleic acid comprising a nucleotide sequence isintroduced into an immune cell in vivo.

In some embodiments, an “immune cell” includes, e.g., a T cell, anatural killer (NK) cell, an NK/T cell, a monocyte, a macrophage, or adendritic cell.

The nucleic acid comprising a nucleotide sequence to be introduced canbe a single bicistronic construct containing an immune activatingreceptor described herein and a target-binding molecule (e.g., scFv)linked to a localizing domain. As described herein, a single bicistronicconstruct can be prepared by inserting an internal ribosomal entry site(IRES) or a 2A peptide-coding region site between the 2 cDNAs encodingthe immune activating receptor as described herein (e.g., CAR) and thetarget-binding molecule (e.g., scFv). The design of tricistronicdelivery systems to delete more than one target should also be feasible.Alternatively, separate transductions (simultaneously or sequentially)of the individual constructs (e.g., CAR and LD-linked target-bindingmolecule) could be performed. Methods of introducing exogenous nucleicacids are exemplified herein, and are well-known in the art.

As used herein, the indefinite articles “a” and “an” should beunderstood to mean “at least one” unless clearly indicated to thecontrary.

EXEMPLIFICATION

Methods

Cloning of scFv from Mouse Anti-Human CD3 Hybridoma

PLU4 hybridoma cells secreting an anti-human CD3 monoclonal antibody(IgG2a isotype; Creative Diagnostics, Shirley, N.Y.) were cultured inIMDM plus GlutaMAX medium (Life Technologies, Carlsbad, Calif.) with 20%fetal bovine serum (Thermo Fisher Scientific, Waltham, Mass.) andantibiotics. Total RNA was extracted using TRIzol reagent (LifeTechnologies), and cDNA was synthesized by M-MLV reverse transcriptase(Promega, Madison, Wis.) and Oligo(dT)₁₅ primer (Promega). IgG LibraryPrimer Set Mouse BioGenomics (US Biological, Salem, Mass.) was used toamplify the variable region of heavy chain (VH) and light chain (VL);PCR products were cloned into TOPO TA cloning kit for sequencing (LifeTechnologies). The VH and VL genes were assembled into scFv by aflexible linker sequence which encodes (Gly₄Ser)₄ using splicing byoverlapping extension-PCR. Signal peptide domain of CD8α was subclonedby PCR using cDNA derived from human activated T cell from healthydonor, and connected to 5′ end of the VL fragment. The Myc tag(EQKLISEEDL; SEQ ID NO: 1) was added to C-terminus of VH by PCR usingsense primer: 5′-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3′ (SEQ ID NO:2) and reverse primer:5′-CAGATCTTCTTCAGAAATAAGTTTTTGTTCGGCTGAGGAGACTGTGAGAG-3′(SEQ ID NO: 3).Also the KDEL (SEQ ID NO: 4) coding sequence was generated after Myc tagby sense primer: 5′-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3′ (SEQ IDNO: 5) and reverse primer:5′-TATATACTCGAGTTACAACTCGTCCTTCAGATCTTCTTCAGAAATAAG-3′ (SEQ ID NO: 6).The synthesized gene consisting of CD8 signal peptide, scFv againsthuman CD3, Myc tag and KDEL (SEQ ID NO: 4) sequence was subcloned intoEcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in whichmyc-KDEL was replaced by other sequences were also made as listed inFIG. 2 .

The sequence of “PEST”-SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ IDNO: 7) motif corresponding to amino acids 422-461 of mouse ornithinedecarboxylase was obtained from GenBank (accession number NM_013614.2).Codon optimization and gene synthesis was done by GenScript (Piscataway,N.J.), and subcloned into the 3′ end of VH by PCR. The constructs weresubcloned into EcoRI and XhoI sites of the MSCV-IRES-GFP vector.

Cloning of scFv Against Human CD7

The sequence scFv derived from murine TH69 (anti-CD7) antibody wasobtained from literature (Peipp et al., Cancer Res 2002 (62):2848-2855). After codon optimization, the synthesized gene consisting ofCD8 signal peptide, scFv against human CD7, Myc tag and KDEL (SEQ ID NO:4) sequence was subcloned into EcoRI and XhoI sites of the MSCV-IRES-GFPvector. Constructs in which myc-KDEL was replaced by other sequenceswere also made as listed in FIG. 2 .

Cloning of scFv Against Human Beta-2 Microglobulin (hB2MG)

The sequence scFv derived from murine BBM.1 (anti-hB2MG) IgG2b antibodywas obtained from literature (Grovender, E. A. et al., Kidney Int. 2004;65(1):310-322). After codon optimization, synthesized gene consists ofCD8 signal peptide, scFv against human B2MG, Myc tag and KDEL (SEQ IDNO: 4) sequence was subcloned into EcoRI and XhoI sites of theMSCV-IRES-GFP vector.

Cloning of scFv Against Human KIR2DL1 and KIR2DL2/DL3

The amino acid sequence of human monoclonal antibody I-7F9(anti-KIR2DL1, KIR2DL2, and KIR2DL3) was derived from publishedInternational Patent Application WO2006003179 A2 by Moretta et al. Aftercodon optimization, the sequence of scFv was designed by connectingvariable light (VL) region and variable heavy (VH) region with linkersequence. The synthesized gene consisting of CD8 signal peptide, scFvagainst human KIRs (KIR2DL1, KIR2DL2 and KIR2DL3), CD8 hinge andtransmembrane domain, and KKMP sequence was subcloned into EcoRI andXhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP wasreplaced by other sequences were also made as listed in FIG. 2 .

Cloning of scFv Against Human NKG2A

The sequence of murine antibody Z199 (anti-NKG2A) was derived from thepublished patent by Spee et al. (EP2247619 A1). After codonoptimization, the sequence of scFv was designed by connecting variablelight (VL) region and variable heavy (VH) region with linker sequence.The synthesized gene consisting of CD8 signal peptide, scFv againsthuman NKG2A, CD8 hinge and transmembrane, and KKMP sequence wassubcloned into EcoRI and XhoI sites of the MSCV-IRES-GFP vector.Constructs in which KKMP was replaced by other sequences were also madeas listed in FIG. 2 . The sequence information for the scFvs generatedherein is shown in Table 1. Sequence information for the variouscomponents depicted in FIG. 2 is shown in Table 2.

Anti-CD19-4-1BB-CD3ζ CAR

This CAR was generated as previously described (Imai, C. et al.,Leukemia. 2004; 18:676-684; Imai, C. et al., Blood. 2005; 106:376-383).

TABLE 1 scFv sequence information Target VH amino acid VL amino acidVH cDNA VL cDNA CD3 EVQLQQSGAELARPGASV QIVLTQSPAIMSASPGAGGTCCAGCTGCAGCAGTCTGGGG CAAATTGTTCTCACCCAGTCTC KMSCKASGYTFTRYTMHWGEKVTMTCSASSSVS CTGAACTGGCAAGACCTGGGGCCTC CAGCAATCATGTCTGCATCTCCVKQRPGQGLEWIGYINPS YMNWYQQKSGTSPKR AGTGAAGATGTCCTGCAAGGCTTCTAGGGGAGAAGGTCACCATGACC RGYTNYNQKFKDKATLTT WIYDTSKLASGVPAHGGCTACACCTTTACTAGGTACACGA TGCAGTGCCAGCTCAAGTGTAA DKSSSTAYMQLSSLTSEDFRGSGSGTSYSLTIS TGCACTGGGTAAAACAGAGGCCTGG GTTACATGAACTGGTACCAGCASAVYYCARYYDDHYCLDY GMEAEDAATYYCQQW ACAGGGTCTGGAATGGATTGGATACGAAGTCAGGCACCTCCCCCAAA WGQGTTLTVSSA SSNPFTFGSGTKLEIATTAATCCTAGCCGTGGTTATACTA AGATGGATTTATGACACATCCA (SEQ ID NO: 12) NRATTACAATCAGAAGTTCAAGGACAA AACTGGCTTCTGGAGTCCCTGC (SEQ ID NO: 13)GGCCACATTGACTACAGACAAATCC TCACTTCAGGGGCAGTGGGTCTTCCAGCACAGCCTACATGCAACTGA GGGACCTCTTACTCTCTCACAAGCAGCCTGACATCTGAGGACTCTGC TCAGCGGCATGGAGGCTGAAGAAGTCTATTACTGTGCAAGATATTAT TGCTGCCACTTATTACTGCCAGGATGATCATTACTGCCTTGACTACT CAGTGGAGTAGTAACCCATTCAGGGGCCAAGGCACCACTCTCACAGT CGTTCGGCTCGGGGACAAAGTT CTCCTCAGCCGGAAATAAACCGG (SEQ ID NO: 14) (SEQ ID NO: 15) CD7 EVQLVESGGGLVKPGGSLAAYKDIQMTQTTSSL GAGGTGCAGCTGGTCGAATCTGGAG GCCGCATACAAGGATATTCAGA (TH69)KLSCAASGLTFSSYAMSW SASLGDRVTISCSAS GAGGACTGGTGAAGCCAGGAGGATCTGACTCAGACCACAAGCTCCCT VRQTPEKRLEWVASISSG QGISNYLNWYQQKPDTCTGAAACTGAGTTGTGCCGCTTCA GAGCGCCTCCCTGGGAGACCGA GFTYYPDSVKGRFTISRDGTVKLLIYYTSSLHS GGCCTGACCTTCTCAAGCTACGCCA GTGACAATCTCTTGCAGTGCATNARNILYLQMSSLRSEDT GVPSRFSGSGSGTDY TGAGCTGGGTGCGACAGACACCTGACACAGGGAATTAGCAACTACCT AMYYCARDEVRGYLDVWG SLTISNLEPEDIATYGAAGCGGCTGGAATGGGTCGCTAGC GAATTGGTATCAGCAGAAGCCA AGTTVTVSSYCQQYSKLPYTFGGG ATCTCCTCTGGCGGGTTCACATACT GATGGCACTGTGAAACTGCTGA(SEQ ID NO: 16) TKLEIKR ATCCAGACTCCGTGAAAGGCAGATT TCTACTATACCTCTAGTCTGCA(SEQ ID NO: 17) TACTATCTCTCGGGATAACGCAAGA CAGTGGGGTCCCCTCACGATTCAATATTCTGTACCTGCAGATGAGTT AGCGGATCCGGCTCTGGGACAGCACTGAGGAGCGAGGACACCGCAAT ACTACAGCCTGACTATCTCCAAGTACTATTGTGCCAGGGACGAAGTG CCTGGAGCCCGAAGATATTGCCCGCGGCTATCTGGATGTCTGGGGAG ACCTACTATTGCCAGCAGTACTCTGGCACTACCGTCACCGTCTCCAG CCAAGCTGCCTTATACCTTTGG CCGGGGGAACAAAGCTGGAGATT (SEQ ID NO: 18) AAAAGG (SEQ ID NO: 19) CD7QVQLQESGAELVKPGASV DIELTQSPATLSVTP CAGGTCCAGCTGCAGGAGTCAGGGGGACATCGAGCTGACACAGTCTC (3a1f) KLSCKASGYTFTSYWMHW GDSVSLSCRASQSISCAGAGCTGGTGAAACCCGGAGCCAG CAGCCACTCTGAGCGTGACCCC VKQRPGQGLEWIGKINPSNNLHWYQQKSHESPR TGTCAAACTGTCCTGTAAGGCCAGC TGGCGATTCTGTCAGTCTGTCANGRTNYNEKFKSKATLTV LLIKSASQSISGIPS GGCTATACTTTCACCAGCTACTGGATGTAGAGCTAGCCAGTCCATCT DKSSSTAYMQLSSLTSED RFSGSGSGTDFTLSITGCACTGGGTGAAACAGAGGCCAGG CTAACAATCTGCACTGGTACCA SAVYYCARGGVYYDLYYYNSVETEDFGMYFCQQ ACAGGGCCTGGAGTGGATCGGCAAG GCAGAAATCACATGAAAGCCCTALDYWGQGTTVTVSS SNSWPYTFGGGTKLE ATTAACCCCAGCAATGGGCGCACCACGGCTGCTGATTAAGAGTGCTT (SEQ ID NO: 20) IKR ACTACAACGAAAAGTTTAAATCCAACACAGAGCATCTCCGGGATTCC (SEQ ID NO: 21) GGCTACACTGACTGTGGACAAGAGCAAGCAGATTCTCTGGCAGTGGG TCCTCTACCGCATACATGCAGCTGA TCAGGAACCGACTTTACACTGTGTTCACTGACATCTGAAGATAGTGC CCATTAACTCTGTGGAGACCGACGTGTACTATTGCGCCAGAGGCGGG AGATTTCGGCATGTATTTTTGCGTCTACTATGACCTGTACTATTACG CAGCAGAGCAATTCCTGGCCTTCACTGGATTATTGGGGGCAGGGAAC ACACATTCGGAGGCGGGACTAA CACAGTGACTGTCAGCTCCACTGGAGATTAAGAGG (SEQ ID NO: 22) (SEQ ID NO: 23) CD45 QVQLVESGGGLVQPGGSLDIVLTQSPASLAVSL CAGGTGCAGCTGGTCGAGTCTGGAG GACATTGTGCTGACCCAGTCCCKLSCAASGFDFSRYWMSW GQRATISCRASKSVS GAGGACTGGTGCAGCCTGGAGGAAGCTGCTTCACTGGCAGTGAGCCT VRQAPGKGLEWIGEINPT TSGYSYLHWYQQKPGTCTGAAGCTGTCATGTGCAGCCAGC GGGACAGAGGGCAACCATCAGC SSTINFTPSLKDKVFISRQPPKLLIYLASNLES GGGTTCGACTTTTCTCGATACTGGA TGCCGAGCCTCTAAGAGTGTCTDNAKNTLYLQMSKVRSED GVPARFSGSGSGTDF TGAGTTGGGTGCGGCAGGCACCAGGCAACAAGCGGATACTCCTATCT TALYYCARGNYYRYGDAM TLNIHPVEEEDAATYAAAAGGACTGGAATGGATCGGCGAG GCACTGGTACCAGCAGAAGCCA DYWGQGTSVTVSYCQHSRELPFTFGSG ATTAACCCAACTAGCTCCACCATCA GGACAGCCACCTAAACTGCTGA(SEQ ID NO: 24) TKLEIK ATTTCACACCCAGCCTGAAGGACAA TCTATCTGGCTTCCAACCTGGA(SEQ ID NO: 25) AGTGTTTATTTCCAGAGATAACGCC ATCTGGAGTGCCTGCACGCTTCAAGAATACTCTGTATCTGCAGATGT TCCGGATCTGGAAGTGGAACCGCCAAAGTCAGGTCTGAAGATACCGC ACTTTACACTGAATATTCACCCCCTGTACTATTGTGCTCGGGGCAAC AGTCGAGGAAGAGGATGCCGCTTACTATAGATACGGGGACGCTATGG ACCTACTATTGCCAGCACAGCCATTATTGGGGGCAGGGAACTAGCGT GGGAGCTGCCCTTCACATTTGG GACCGTGAGTCAGCGGGACTAAGCTGGAGATC (SEQ ID NO: 26) AAG (SEQ ID NO: 27) B2MGEVQLQQSGAELVKPGASV DIQMTQSPASQSASL GAGGTGCAGCTGCAGCAGAGCGGAGGATATTCAGATGACCCAGTCCC KLSCTPSGFNVKDTYIHW GESVTITCLASQTIGCAGAACTGGTGAAACCTGGAGCCAG CTGCATCACAGAGCGCCTCCCT VKQRPKQGLEWIGRIDPSTWLAWYQQKPGKSPQ CGTCAAGCTGTCCTGTACTCCATCT GGGCGAGTCAGTGACCATCACADGDIKYDPKFQGKATITA LLIYAATSLADGVPS GGCTTCAACGTGAAGGACACATACATGCCTGGCTAGCCAGACAATTG DTSSNTVSLQLSSLTSED RFSGSGSGTKFSLKITTCACTGGGTCAAGCAGCGGCCCAA GCACTTGGCTGGCATGGTACCA TAVYYCARWFGDYGAMNYRTLQAEDFVSYYCQQ ACAGGGACTGGAGTGGATCGGCAGA GCAGAAGCCCGGCAAATCCCCTWGQGTSVTVSS LYSKPYTFGGGTKLE ATTGACCCATCCGACGGCGATATCACAGCTGCTGATCTATGCAGCTA (SEQ ID NO: 28) IKRAD AGTATGATCCCAAATTCCAGGGGAACCTCTCTGGCAGACGGAGTGCC (SEQ ID NO: 29) GGCTACTATTACCGCAGATACCAGCCAGTAGGTTCTCTGGGAGTGGA TCCAACACAGTGAGTCTGCAGCTGT TCAGGCACCAAGTTTTCTCTGACTAGTCTGACTAGCGAAGACACCGC AAATTCGCACACTGCAGGCTGACGTCTACTATTGTGCTAGATGGTTT GGATTTCGTCTCCTACTATTGCGGCGATTACGGGGCCATGAATTATT CAGCAGCTGTACTCTAAACCTTGGGGGCAGGGAACCAGCGTCACCGT ATACATTTGGCGGGGGAACTAA GTCCAGCGCTGGAAATCAAACGAGCAGAC (SEQ ID NO: 30) (SEQ ID NO: 31) NKG2AEVQLVESGGGLVKPGGSL QIVLTQSPALMSASP GAGGTGCAGCTGGTGGAGAGCGGAGCAGATTGTCCTGACCCAGTCTC KLSCAASGFTFSSYAMSW GEKVTMTCSASSSVSGAGGACTGGTGAAGCCAGGAGGAAG CAGCCCTGATGAGCGCCTCCCC VRQSPEKRLEWVAEISSGYIYWYQQKPRSSPKP CCTGAAGCTGTCCTGTGCCGCCTCT TGGCGAGAAGGTGACAATGACCGSYTYYPDTVTGRFTISR WIYLTSNLASGVPAR GGCTTCACATTTTCCTCTTATGCAATGCTCTGCCAGCTCCTCTGTGA DNAKNTLYLEISSLRSED FSGSGSGTSYSLTISTGAGCTGGGTGCGGCAGTCCCCAGA GCTACATCTATTGGTACCAGCA TAMYYCTRHGDYPRFFDVSMEAEDAATYYCQQW GAAGAGACTGGAGTGGGTGGCAGAG GAAGCCTCGGAGCTCCCCAAAGWGAGTTVTVSS SGNPYTFGGGTKLEI ATCAGCTCCGGAGGATCCTACACCTCCCTGGATCTATCTGACATCCA (SEQ ID NO: 32) KR ACTATCCTGACACAGTGACCGGCCGACCTGGCCTCTGGCGTGCCAGC (SEQ ID NO: 33) GTTCACAATCTCTAGAGATAACGCCCAGATTCTCTGGCAGCGGCTCC AAGAATACCCTGTATCTGGAGATCT GGCACATCTTACAGCCTGACCACTAGCCTGAGATCCGAGGATACAGC TCTCTAGCATGGAGGCCGAGGACATGTACTATTGCACCAGGCACGGC CGCCGCCACCTACTATTGCCAGGACTACCCACGCTTCTTTGACGTGT CAGTGGTCCGGCAATCCATATAGGGGAGCAGGAACCACAGTGACCGT CATTTGGCGGCGGCACCAAGCT GTCCTCT GGAGATCAAGAGG(SEQ ID NO: 34) (SEQ ID NO: 35) KIR QVQLVQSGAEVKKPGSSV EIVLTQSPVTLSLSPCAGGTCCAGCTGGTGCAGTCTGGAG GAGATCGTGCTGACCCAGTCTC 2DL1 KVSCKASGGTFSFYAISWGERATLSCRASQSVS CTGAAGTGAAGAAACCAGGGAGCTC CTGTCACACTGAGTCTGTCACC and 2/3VRQAPGQGLEWMGGFIPI SYLAWYQQKPGQAPR CGTCAAGGTGTCATGCAAAGCAAGCAGGGGAACGGGCTACACTGTCT FGAANYAQKPQGRVTITA LLIYDASNRATGIPAGGCGGGACTTTCTCCTTTTATGCAA TGCAGAGCAAGCCAGTCCGTGA DESTSTAYMELSSLRSDDRFSGSGSGTDFTLTI TCTCTTGGGTGAGACAGGCACCTGG GCTCCTACCTGGCCTGGTATCATAVYYCARIPSGSYYYDY SSLEPEDFAVYYCQQ ACAGGGACTGGAGTGGATGGGAGGCGCAGAAGCCAGGCCAGGCTCCC DMDVWGQGTTVTVSS RSNWMYTFGQGTKLETTCATCCCAATTTTTGGAGCCGCTA AGGCTGCTGATCTACGATGCAA (SEQ ID NO: 36) IKRTACTATGCCCAGAAGTTCCAGGGCAG GCAACAGGGCCACTGGGATTCC (SEQ ID NO: 37)GGTGACCATCACAGCTGATGAGTCT CGCCCGCTTCTCTGGCAGTGGGACTAGTACCGCATACATGGAACTGT TCAGGAACCGACTTTACTCTGACTAGTCTGAGGAGCGACGATACCGC CCATTTCTAGTCTGGAGCCTGACGTGTACTATTGTGCTCGCATTCCA AGATTTCGCCGTGTACTATTGCTCAGGCAGCTACTATTACGACTATG CAGCAGCGATCCAATTGGATGTATATGGACGTGTGGGGCCAGGGGAC ATACTTTTGGCCAGGGGACCAA CACAGTCACCGTGAGCAGCGCTGGAGATCAAACGGACA (SEQ ID NO: 39) (SEQ ID NO: 38)

TABLE 2 Sequence information for components depicted in FIG. 2 CD8 SPVH-VL VH-VL amino linker CD 8 hinge and linker CD 8 hinge and Targetacid amino acid TM amino acid CD8 SP cDNA cDNA TM cDNA CD3 MALPVTAGGGGSGGGGS KPTTTPAPRPPTPA ATGGCCTTACC GGTGGTGGTG AAGCCCACCACGAC LLLPLALGGGGSGGGGS PTIASQPLSLRPEA AGTGACCGCCT GTTCTGGTGG GCCAGCGCCGCGAC LLHAARP(SEQ ID CRPAAGGAVHTRGL TGCTCCTGCCG TGGTGGTTCT CACCAACACCGGCG (SEQ IDNO: 41) DFACDIYIWAPLAG CTGGCCTTGCT GGCGGCGGCG CCCACCATCGCGTC NO: 40)TCGVLLLSLVITLY GCTCCACGCCG GCTCCGGTGG GCAGCCCCTGTCCC (SEQ ID CCAGGCCGTGGTGGATCC TGCGCCCAGAGGCG NO: 42) (SEQ ID (SEQ ID TGCCGGCCAGCGGC NO: 44)NO: 51) GGGGGGCGCAGTGC ACACGAGGGGGCTG GACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGG ACTTGTGGGGTCCT TCTCCTGTCACTGG TTATCACCCTTTAC(SEQ ID NO: 57) CD7 MALPVTA GGGGSGGGGS TTTPAPRPPTPAPT ATGGCTCTGCCGGAGGAGGAG ACCACTACACCTGC (TH69) LLLPLAL GGGGSGGGGS IASQPLSLRPEACRTGTGACCGCAC GAAGCGGAGG ACCAAGGCCTCCCA LLHAARP (SEQ ID PAAGGAVHTRGLDFTGCTGCTGCCC AGGAGGATCC CACCCGCTCCCACT (SEQ ID NO: 41) ACDIYIWAPLAGTCCTGGCTCTGCT GGAGGCGGGG ATCGCTTCCCAGCC NO: 40) GVLLLSLVITLY GCTGCACGCCGGATCTGGAGG ACTGTCCCTGAGGC (SEQ ID CAAGACCT AGGAGGAAGT CCGAGGCCTGCAGGNO: 50) (SEQ ID (SEQ ID CCAGCAGCTGGCGG NO: 45) NO: 52) AGCCGTGCATACTAGGGGGCTGGACTTC GCTTGCGACATCTA CATCTGGGCCCCAC TGGCAGGGACATGCGGAGTCCTGCTGCT GTCCCTGGTCATCA CACTTTAC (SEQ ID NO: 58) CD7 MALPVTAGGGGSGGGGS TTTPAPRPPTPAPT ATGGCTCTGCC GGAGGAGGAG ACTACCACACCAGC (3a1f)LLLPLAL GGGGS IASQPLSLRPEACR CGTCACCGCTC GATCCGGCGG TCCAAGACCACCTALLHAARP (SEQ ID PAAGGAVHTRGLDF TGCTGCTGCCT AGGAGGCTCT CCCCTGCACCAACA(SEQ ID NO: 43) ACDIYIWAPLAGTC CTGGCTCTGCT GGGGGAGGCG ATTGCTAGTCAGCCNO: 40) GVLLLSLVITLY GCTGCACGCTG GGAGT(SEQ ACTGTCACTGAGAC (SEQ IDCTCGACCA ID NO: 53) CAGAAGCATGTAGG NO: 50) (SEQ ID CCTGCAGCTGGAGGNO: 46) AGCTGTGCACACCA GAGGCCTGGACTTT GCCTGCGATATCTA CATTTGGGCTCCTCTGGCAGGAACCTGT GGCGTGCTGCTGCT GTCTCTGGTCATCA CACTTTAC (SEQ ID NO: 59)CD45 MALPVTA GGGGSGGGGS KPTTTPAPRPPTPA ATGGCTCTGCC GGAGGAGGAGAAGCCCACCACGAC LLLPLAL GGGGSGGGGS PTIASQPLSLRPEA CGTGACCGCTC GAAGTGGAGGGCCAGCGCCGCGAC LLHAARP (SEQ ID CRPAAGGAVHTRGL TGCTGCTGCCT AGGAGGATCACACCAACACCGGCG (SEQ ID NO: 41) DFACDIYIWAPLAG CTGGCTCTGCT GGAGGCGGGGCCCACCATCGCGTC NO: 40) TCGVLLLSLVITLY GCTGCATGCTG GAAGCGGCGGGCAGCCCCTGTCCC (SEQ ID CTCGACCT GGGAGGCTCC TGCGCCCAGAGGCG NO: 42)(SEQ ID (SEQ ID TGCCGGCCAGCGGC NO: 47) NO: 54) GGGGGGCGCAGTGCACACGAGGGGGCTG GACTTCGCCTGTGA TATCTACATCTGGG CGCCCTTGGCCGGGACTTGTGGGGTCCT TCTCCTGTCACTGG TTATCACCCTTTAC (SEQ ID NO: 57) B2MGMALPVTA GGGGSGGGGS KPTTTPAPRPPTPA ATGGCCCTGCC GGAGGAGGAG AAGCCCACCACGACLLLPLAL GGGGSGGGGS PTIASQPLSLRPEA CGTCACCGCCC GAAGTGGAGG GCCAGCGCCGCGACLLHAARP (SEQ ID CRPAAGGAVHTRGL TGCTGCTGCCC AGGAGGGTCA CACCAACACCGGCG(SEQ ID NO: 41) DFACDIYIWAPLAG CTGGCTCTGCT GGAGGCGGGG CCCACCATCGCGTCNO: 40) TCGVLLLSLVITLY GCTGCACGCCG GAAGCGGCGG GCAGCCCCTGTCCC (SEQ IDCAAGACCC GGGAGGATCC TGCGCCCAGAGGCG NO: 42) (SEQ ID (SEQ IDTGCCGGCCAGCGGC NO: 48) NO: 55) GGGGGGCGCAGTGC ACACGAGGGGGCTGGACTTCGCCTGTGA TATCTACATCTGGG CGCCCTTGGCCGGG ACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACCCTTTAC (SEQ ID NO: 57) NKG2A MALPVTA GGGGSGGGGSKPTTTPAPRPPTPA ATGGCTCTGCC GGAGGAGGAG AAGCCAACCACAAC LLLPLAL GGGGSGGGGSPTIASQPLSLRPEA CGTGACCGCCC GATCTGGAGG CCCTGCACCAAGGC LLHAARP (SEQ IDCRPAAGGAVHTRGL TGCTGCTGCCT AGGAGGCAGC CACCTACACCAGCA (SEQ ID NO: 41)DFACDIYIWAPLAG CTGGCTCTGCT GGCGGCGGCG CCTACCATCGCAAG NO: 40)TCGVLLLSLVITLY GCTGCACGCTG GCTCCGGCGG CCAGCCACTGTCCC (SEQ ID CCCGCCCACGGCGGCTCT TGAGGCCAGAGGCA NO: 42) (SEQ ID (SEQ ID TGTAGGCCTGCAGC NO: 49)NO: 56) AGGAGGCGCCGTGC ACACACGCGGCCTG GACTTTGCCTGCGA TATCTACATCTGGGCACCACTGGCAGGA ACCTGTGGCGTGCT GCTGCTGAGCCTGG TGATTACCCTGTAT(SEQ ID NO: 60) KIR MALPVTA GGGGSGGGGS KPTTTPAPRPPTPA ATGGCCTTACCGGTGGTGGTG AAGCCCACCACGAC 2DL1 LLLPLAL GGGGSGGGGS PTIASQPLSLRPEAAGTGACCGCCT GTTCTGGTGG GCCAGCGCCGCGAC and 2/3 LLHAARP (SEQ IDCRPAAGGAVHTRGL TGCTCCTGCCG TGGTGGTTCT CACCAACACCGGCG (SEQ ID NO: 41)DFACDIYIWAPLAG CTGGCCTTGCT GGCGGCGGCG CCCACCATCGCGTC NO: 40)TCGVLLLSLVITLY GCTCCACGCCG GCTCCGGTGG GCAGCCCCTGTCCC (SEQ ID CCAGGCCGTGGTGGATCC TGCGCCCAGAGGCG NO: 42) (SEQ ID (SEQ ID TGCCGGCCAGCGGC NO: 44)NO: 51) GGGGGGCGCAGTGC ACACGAGGGGGCTG GACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGG ACTTGTGGGGTCCT TCTCCTGTCACTGG TTATCACCCTTTAC(SEQ ID NO: 57)

Gene Transduction, Cell Expansion, Flow Cytometric Analysis andFunctional Studies

These were performed as previously described (Kudo, K et al., CancerRes. 2014; 74(1):93-103).

Results

Generation of scFv Constructs

A schematic of the technology is outlined in FIG. 1 . A schematicrepresentation of the inhibitory constructs that we generated is shownin FIG. 2 . The scFv portion can be derived by cloning the cDNA encodingvariable light (VL) and variable heavy (VH) immunoglobulin chain regionsfrom an antibody-producing hybridoma cell line or from the correspondingpublished sequences. VL and VH are linked with a short peptide sequence(“linker”) according to standard techniques to make a full scFv. To beexpressed, the scFv is linked to a signal peptide at the N-terminus; thesignal peptide is required for the scFv to be expressed, as confirmed inpreliminary experiments. Proteins containing scFv plus signal peptideare generally released into the cells' milieu. For example, inpreliminary experiments (not shown), an anti-CD3ε scFv plus signalpeptide expressed in Jurkat T cells was detected in the cells' culturesupernatant. By directing the scFv to specific compartments andpreventing its secretion, possible effects on other cells are prevented.To direct it to the endoplasmic reticulum (ER), the KDEL (SEQ ID NO: 4)motif (which retains proteins in the ER) was utilized (Strebe N. et al.,J Immunol Methods. 2009; 341(1-2):30-40). To promote the degradation ofthe targeted protein, we linked it to a proteasome-targeting PEST motif(Joshi, S. N. et al., MAbs. 2012; 4(6):686-693). The scFv can also bedirected to the cell membrane by linking it to the transmembrane domainand hinge of CD8a or another transmembrane protein.

Downregulation of T-Cell Receptor in T Lymphocytes ExpressingAnti-CD19-BB-ζ CAR

To determine whether the proposed strategy could be applied to generateimmune cells expressing CAR and lacking one or more markers, T-cellreceptor (TCR) expression was downregulated in anti-CD19 CAR T-cells.

To be expressed on the cell membrane, the CD3/TCR complex requiresassembly of all its components (TCRα, TCRβ, CD3δ, CD3ζ, CD3γ, CD3ζ).Lack of one component prevents CD3/TCR expression and, therefore,antigen recognition. In preliminary studies, the scFv from an anti-CD3εhybridoma (purchased from Creative Diagnostics, Shirley, N.Y.) wascloned and generated the constructs containing KDEL (SEQ ID NO: 4),PEST, CD8a transmembrane domain or others as shown in FIG. 2 .

The constructs disclosed herein were transduced in the CD3/TCR+ Jurkatcell line using a murine stem cell virus (MSCV) retroviral vectorcontaining green fluorescent protein (GFP). Percentage of GFP+ cellsafter transduction was >90% in all experiments. FIG. 3A shows results ofstaining with anti-CD3ε antibody among GFP+ cells, as measured by flowcytometry. Antibody staining of CD3ε was decreased to variable degree incells transduced with the constructs listed. Similar downregulation ofCD3ε was obtained with human peripheral blood T lymphocytes (FIG. 3B).FIG. 3C shows illustrative flow cytometry dot plots of CD3ε expressionin GFP-positive Jurkat cells after transduction with different geneconstructs in comparison with cells transduced with a vector containingGFP alone. Downregulation of CD3 did not affect growth of Jurkat cellsor expression of all other cell markers tested, including CD2, CD4, CD8,CD45, CD25, CD69. Inhibition of CD3 expression persisted for over 3months. Further enrichment of CD3-negative cells could be achieved byCD3+ T cell depletion with anti-CD3 magnetic beads (Dynal, LifeTechnologies, Carlsbad, Calif.).

Staining with anti-TCRαβ antibody of Jurkat cells or human peripheralblood T lymphocytes showed that down regulation of CD3ε expression wasassociated with dowregulation of TCRαβ expression (FIG. 4 ).

Next, it was determined whether the anti-CD3 scFv-myc KDEL could beexpressed simultaneously with an anti-CD19-4-1BB-CD3ζ CAR. As shown inFIG. 5 , this resulted in T cells lacking CD3 expression whileexpressing the anti-CD19 CAR. TCR was also absent on these cells (notshown).

To assess whether CAR could signal in Jurkat cells with downregulatedCD3/TCR, the expression of the activation markers CD69 and CD25 wastested, and exocytosis of lytic granules was measured by CD107aexpression in Jurkat cells co-cultured with the CD19+ leukemia cell lineOP-1. As shown in FIG. 6 , downregulation of CD3/TCR with the anti-CD3scFv-myc KDEL construct did not diminish the capacity ofanti-CD19-4-1BB-CD3ζ CAR to activate Jurkat cells. To further explorethe effects of CD3/TCR deletion on CAR signaling, it was determinedwhether CD3-negative T lymphocytes expressing the CAR could bestimulated by its ligation. As shown in FIG. 7 , co-culture of Tlymphocytes expressing the anti-CD19 CAR with CD19+ leukemic cells ledto T cell proliferation regardless of whether CD3 was downregulated ornot, indicating that CD3/TCR downregulation did not diminish the CARproliferative stimulus.

Accordingly, CD3/TCR can be effectively downregulated in CAR-T cellsusing the anti-CD3 scFv-myc KDEL construct without affecting T cellactivation, degranulation and proliferation driven by the CAR.

Downregulation of CD7

It was determined whether the strategy that successfully modulatedCD3/TCR expression could be applied to other surface molecules. For thispurpose, CD7 expression was modulated. The scFv sequence was derivedfrom that published by Peipp et al. (Cancer Res 2002 (62): 2848-2855),which was linked to the CD8 signal peptide and the myc-KDEL sequence asillustrated in FIG. 2 . Using the MSCV retroviral vector, theanti-CD7-myc KDEL construct was transduced in peripheral bloodlymphocytes, which have high expression of CD7 as detected by ananti-CD7 antibody conjugated to phycoerythrin (BD Bioscience). As shownin FIG. 8 , CD7 in T lymphocytes transduced with the construct wasvirtually abrogated.

Downregulation of HLA-Class I

The strategy was then applied to downregulate another surface molecule,HLA class I.

HLA class I consists of polymorphic a chains and a non-polymorphic chaintermed β2-microglobulin. Knock-down of the latter subunit results inabrogation of HLA (MHC in the mouse) Class I expression (Koller, B H etal., Science. 1990; 248(4960):1227-1230). An scFv reacting withβ2-microglobulin was used to suppress expression of HLA Class I inimmune cells.

The scFv sequence was derived from that published by Grovender et al.(Kidney Int. 2004; 65(1):310-322), which was linked to the CD8 signalpeptide and the myc KDEL sequence as illustrated in FIG. 2 . Using theMSCV retroviral vector, the anti-β2M-myc KDEL construct was transducedin Jurkat cells, which have high expression of HLA Class I as detectedby an anti-HLA-ABC antibody conjugated to phycoerythrin (BD Pharmingen).As shown in FIG. 9 , Jurkat cells transduced with the construct had asubstantial downregulation of HLA-ABC expression. Cells maintained theirmorphology and growth capacity.

Dowregulation of Inhibitory Receptors in NK Cells

To determine if the strategy outlined above would also apply to surfacemolecules expressed in other immune cells, downregulation of function ofthe inhibitory receptor KIR2DL1, KIR2DL2/DL3 and NKG2A was tested in NKcells.

To downregulate MR receptors, an scFv reacting with KIR2DL1 andKIR2DL2/DL3 was used to suppress their expression in NK cells. The scFvsequence was derived from that published by Moretta et al. (patentWO2006003179 A2), which was linked to the CD8 signal peptide and the ERretention sequences as illustrated in FIG. 2 . Using the MSCV retroviralvector, the constructs were transduced in NK cells expanded from humanperipheral blood and selected for KIR2DL1 expression. These cells hadhigh KIR2DL1 expression as detected by an anti-KIR2DL1 antibodyconjugated to allophycocyanin (R&D Systems) and also high KIR2DL2/DL3expression as detected by an anti-KIR2DL2/DL3 antibody conjugated tophycoerythrin (BD Bioscience). FIG. 10 shows results obtained withscFv-linker(20) AEKEDL and scFv-EEKKMP, with substantial down regulationof the targeted KIRs.

To downregulate NKG2A, an scFv reacting with NKG2A was used to suppressits expression in NK cells. The scFv sequence, which was derived frompublished European Patent Application No. EP2247619 A1 by Spee et al.was linked to the CD8 signal peptide and the ER retention sequences asillustrated in FIG. 2 . Using the MSCV retroviral vector, the constructswere transduced in NK cells expanded from human peripheral blood, whichhad high NKG2A expression as detected by an anti-NKG2A antibodyconjugated to phycoerythrin (Beckman Coulter). FIG. 11 shows substantialdownregulation of NKG2A obtained with scFv-EEKKMP.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An isolated engineered immune cell expressing achimeric antigen receptor (CAR) at the cell surface, wherein theengineered immune cell comprises a polynucleotide encoding a CARcomprising a CD7 binding domain, a transmembrane domain, at least oneco-stimulatory domain, and a signaling domain, wherein the CD7 bindingdomain comprises a single chain variable fragment (scFv) that bindsspecifically to CD7 comprising: a) a variable heavy chain sequencehaving at least 95% sequence identity to SEQ ID NO: 16, comprising: aheavy chain (HC) complementarity determining region (CDR) 1 comprisingamino acids 26-32 of SEQ ID NO: 16, a HC CDR2 comprising amino acids52-58 of SEQ ID NO: 16, and a HC CDR3 comprising amino acids 98-106 ofSEQ ID NO: 16, and a variable light chain sequence having at least 95%sequence identity to SEQ ID NO: 17, comprising: a light chain (LC) CDR1comprising amino acids 28-38 of SEQ ID NO: 17, a LC CDR2 comprisingamino acids 54-60 of SEQ ID NO: 17, and a LC CDR3 comprising amino acids93-101 of SEQ ID NO: 17; or b) a variable heavy chain sequence having atleast 95% sequence identity to SEQ ID NO: 20, comprising: a HC CDR1comprising amino acids 26-32 of SEQ ID NO: 20, a HC CDR2 comprisingamino acids 52-57 of SEQ ID NO: 20, and a HC CDR3 comprising amino acids99-112 of SEQ ID NO: 20, and a variable light chain sequence having atleast 95% sequence identity to SEQ ID NO: 21, comprising: a LC CDR1comprising amino acids 24-34 of SEQ ID NO: 21, a LC CDR2 comprisingamino acids 50-56 of SEQ ID NO: 21, and a LC CDR3 comprising amino acids89-97 of SEQ ID NO: 21; wherein the signaling domain is a CD3ζ signalingdomain, and wherein the costimulatory domain is a 4-1BB costimulatorydomain or a CD28 costimulatory domain.
 2. The isolated engineered immunecell of claim 1, wherein the engineered immune cell is an engineered Tcell or an engineered NK cell.
 3. The isolated engineered immune cell ofclaim 1, further comprising a polynucleotide encoding a polypeptide thatdownregulates endogenous CD7 surface expression, wherein the polypeptidethat downregulates endogenous CD7 surface expression comprises a secondCD7 binding domain comprising the HC CDR1, the HC CDR2, the HC CDR3, theLC CDR1, the LC CDR2, and the LC CDR3 of the CD7 binding domain of theCAR linked to a localization domain, wherein the localization domaincomprising an ER retention signal, a Golgi retention signal, or aproteosome localization sequence, wherein if the ER retention signalcomprises the amino acid sequence KDEL, the polypeptide thatdownregulates endogenous CD7 surface expression further comprises alinker sequence between the second CD7 binding domain and thelocalization domain.
 4. The isolated engineered immune cell of claim 3,wherein the ER retention sequence comprises the amino acid sequenceKDEL, KKXX, or KXD/E, wherein X is any amino acid.
 5. The isolatedengineered immune cell of claim 4, wherein the engineered immune cell isan engineered T cell or an engineered NK cell.
 6. The isolatedengineered immune cell of claim 3, wherein the Golgi retention signalcomprises the amino acid sequence YQRL or the proteosome localizationsequence comprises a PEST motif.
 7. An isolated engineered immune cellcomprising a polynucleotide encoding an engineered polypeptidecomprising a CD7 binding domain linked to a localization domain thatdownregulates endogenous CD7 surface expression, wherein the CD7 bindingdomain comprises a single chain variable fragment (scFv) that bindsspecifically to CD7 comprising: a) a variable heavy chain sequencehaving at least 95% sequence identity to SEQ ID NO: 16, comprising: aheavy chain (HC) complementarity determining region (CDR) 1 comprisingamino acids 26-32 of SEQ ID NO: 16, a HC CDR2 comprising amino acids52-58 of SEQ ID NO: 16, and a HC CDR3 comprising amino acids 98-106 ofSEQ ID NO: 16, and a variable light chain sequence having at least 95%sequence identity to SEQ ID NO: 17, comprising: a light chain (LC) CDR1comprising amino acids 28-38 of SEQ ID NO: 17, a LC CDR2 comprisingamino acids 54-60 of SEQ ID NO: 17, and a LC CDR3 comprising amino acids93-101 of SEQ ID NO: 17; or b) a variable heavy chain sequence having atleast 95% sequence identity to SEQ ID NO: 20, comprising: a HC CDR1comprising amino acids 26-32 of SEQ ID NO: 20, a HC CDR2 comprisingamino acids 52-57 of SEQ ID NO: 20, and a HC CDR3 comprising amino acids99-112 of SEQ ID NO: 20, and a variable light chain sequence having atleast 95% sequence identity to SEQ ID NO: 21, comprising: a LC CDR1comprising amino acids 24-34 of SEQ ID NO: 21, a LC CDR2 comprisingamino acids 50-56 of SEQ ID NO: 21, and a LC CDR3 comprising amino acids89-97 of SEQ ID NO: 21; wherein the localization domain comprising an ERretention signal, a Golgi retention signal, or a proteosome localizationsequence; and wherein if the ER retention signal comprises the aminoacid sequence KDEL, the polypeptide that downregulates endogenous CD7surface expression further comprises a linker sequence between the CD7binding domain and the localization domain.
 8. The isolated engineeredimmune cell of claim 7, wherein the engineered immune cell is anengineered T cell or an engineered NK cell.
 9. The isolated engineeredimmune cell of claim 7, wherein the ER retention signal comprises theamino acid sequence KDEL.
 10. The isolated engineered immune cell ofclaim 8, wherein the ER retention sequence comprises the amino acidsequence KKXX, or KXD/E, wherein X is any amino acid.
 11. The isolatedengineered immune cell of claim 9, wherein the engineered immune cell isan engineered T cell or an engineered NK cell.
 12. The isolatedengineered T cell or NK cell of claim 8, wherein the Golgi retentionsignal comprises the amino acid sequence YQRL or the proteosomelocalization sequence comprises a PEST motif.